Since its inception in 1962, R. B. Merrifield's concept of solid phase peptide synthesis has seen many improvements and has now become an established technique in the art. Literally hundreds of investigations have been published describing the chemical details of the method (See for example, Merrifield, R. B.: Science 150, 178 (1965); Merrifield, R. B.: Sci. Amer. 218, 56 (1968); Stewart, J. M., Young, J. D.: In: Solid Phase Peptide Synthesis. San Francisco, Calif.: Freeman 1969; and Erickson, B. W., Merrifield, R. B.: In: The Proteins (eds. Neurath, R. L. Hill), III. Ed., Vol. 2, pp 255-527. New York: Academic Press 1976).)
Typically, solid phase peptide synthesis begins with the covalent attachment of the carboxyl end of an (alpha-amino protected) first amino acid in the peptide sequence through an organic linker to an insoluble resin bead (typically 25-300 microns in diameter), illustrated by: ##STR1##
A general cycle of synthesis then consists of deprotection of the resin bound alpha-amino group, washing (and neutralization if necessary), followed by reaction with with some carboxyl activated form of the next (alpha-amino protected) amino acid to yield: ##STR2## Repetition of the cycle to the n.sup.th amino acid then yields: ##STR3## At the end of the synthesis, the link of the peptide to its polymer support is cleaved, and the dissolved peptide is separated from the insoluble resin and purified.
Although this process is simple in principle, in practice it can be quite difficult to obtain peptides over about 30 amino acids long which have any substantial purity. The reason for this is that the average step yield has a profound effect on the purity of the product peptide, as illustrated by the values in the following table for synthesis of a 30 amino acid peptide.
TABLE ______________________________________ 30 AMINO ACID PEPTIDE Average Step Yield (%) Product Purity (%) ______________________________________ 95.0 21 99.5 86 99.7 91 ______________________________________
The results are even more problematic for longer peptides, eg. for a target peptide with 101 residues, a step yield of 99.0% provides a product of only 36% purity. In all cases, the by-products of peptide synthesis consist of a complex mixture of molecules which are chemically similar to the target peptide. Chromatographic purification can be extraordinarily difficult and time consuming as the relative amount of by-product molecules begins to exceed about 25%.
The efficiency of step yield is dependent on many factors such as the nature and quality of the protected amino acids, solvent purity, chemical integrity of the resin, the chemical nature of the organic linker, the form of the activated carboxyl of the amino acid, efficiency of the wash steps, the synthesis protocol, and in some instances the identity of an amino acid in conjunction with a particular sequence segment to which it is being added.
Each of the above factors, when not optimally controlled, will contribute some significant increment to yield reduction in every coupling step. At the present time, the complexity of these factors is such that average step yields in solid phase peptide synthesis are typically in the range of 93-97% for both manual and automated executions. For practical applications on a commercially reasonable scale, such as for the development of pharmaceuticals, enzyme substrates and inhibitors, hormones, vaccines, and diagnostic reagents, such low step yields significantly increase costs of production and in many cases make such direct solid phase synthesis of peptides impractical.
Prior art peptide synthesizers operate essentially as "washing machines" which automate the monotonous fluid manipulations of deprotection, addition of coupling agent, and washing. In no case do existing commercial peptide synthesizers form an activated amino acid species outside or independent of the reaction vessel. Typically, protected amino acid and DCC are added to the reaction vessel containing the resin bound, incipient peptide chain so that activation of the amino acid occurs in the presence of the deprotected alpha-amino group. This approach both limits the possibility (or feasibility) of optimizing activation conditions for individual amino acids and requires that any modification or activation conditions be done in the presence of the deprotected alpha-amino group and the growing, resin-bound peptide chain. This fact makes it difficult, if not impossible, to optimize activation parameters by analyzing rates of formation and relative thermal and solvent stabilities of the individual, activated amino acid species. Additionally, the ability to use various thermal inputs during the activation process can only be done in the presence of the peptide chain.